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Antioxidants determination with laccase


Juozas Kulys a,b,∗ , Irina Bratkovskaja a
aInstitute of Biochemistry, Department of Enzyme Chemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania
b Vilnius Gediminas Technical University, Faculty of Fundamental Sciences, Department of Chemistry and Bioengineering,
Sauletekio Avenue 11, LT-10223 Vilnius, Lithuania
Received 16 August 2006; received in revised form 5 November 2006; accepted 8 November 2006

Abstract
The spectrophotometric method of antioxidants determination using recombinant laccase Polyporus pinsitus (rPpL) and Myceliophthora ther-
mophila (rMtL) was developed. The method includes simultaneous oxidation of the antioxidant and high reactive laccase substrate producing
chromophoric radical cation. As laccase substrates ABTS and other high reactive phenoxazine derivatives: 2-phenoxazin-10-yl-ethanol (PET),
3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) and 3-phenoxazin-10-yl-propionic acid (PPA) were used. The kinetic data were analysed
using a scheme of simultaneous oxidation of the antioxidant and the substrate.
In a range of (0.9–7.3) × 10−6 M of Trolox the measurings recovered 91 and 99% of the antioxidant if ABTS and both laccases were used. The
recovery varied between 82 and 124% if phenoxazine derivatives were used. The antioxidant activity determined in rich with antioxidants food
samples, i.e. date-palm, black raisin, golden raisin, skin of red grape, dice of red grape, fitted the literature data.
© 2006 Published by Elsevier B.V.

Keywords: Antioxidant; Trolox; Laccase; ABTS; Phenoxazine; Radical cation

1. Introduction electrophoresis [3]. However, the information about the number


and the concentration of antioxidants present in the sample
The exposure of living organisms to reactive oxygen, is not sufficient to obtain biologically relevant information
nitrogen, chlorine, and bromine species (RSs) is common in about the antioxidant activity of the sample, primarily due
aerobic life [1,2]. RSs fall into two groups, i.e. those that contain to the synergistic interaction between different antioxidants.
unpaired electrons (O2 •− , OH• , NO• ), and those that have the For this reason methods for measuring the overall antioxidant
ability to extract electrons from other molecules (H2 O2 , HOCl, activity have been introduced which provide the parameter of
and HOBr). These species may damage biomolecules directly, the cumulative action of all antioxidants present in the sample.
or initiate chain reactions resulting in extensive damage of Two types of analytical methods are currently used for
cell structures. It was recognized that natural antioxidants, evaluation of the antioxidant activity [3]: (i) inhibition meth-
due to their RS scavenging activity, might have beneficial ods, in which the inhibition of oxidative damage of the
effects in protection against these damages. Consequently, target molecule is measured in the presence of antioxidants
considerable interest has been focused on the analytical meth- and related to a known standard, and (ii) methods based
ods for determination of antioxidants in biological samples. on direct measurement of scavenging stable free radicals
Determination of antioxidants in the biological samples usually by antioxidants present in the sample. The most popular
requires the use of high-resolving analytical techniques, such scavenging method is 6-hydroxy-2,5,7,8-tetramethylchroman-
as high-performance liquid chromatography and capillary 2-carboxylic acid (Trolox) equivalent antioxidant capacity
(TE) decolourisation assay based on the scavenging of stable
2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical
∗ Corresponding author at: Vilnius Gediminas Technical University, Fac- cation (ABTS•+ ) [4–6] or 2,2-diphenyl-1-picrylhydrazyl radical
ulty of Fundamental Sciences, Department of Chemistry and Bioengineering,
Sauletekio Avenue 11, LT-10223 Vilnius, Lithuania. Tel. +370 5 2729176;
(DPPH• ) [7]. The ABTS•+ typically is prepared under action of
fax: +370 5 2729196. peroxidase or myoglobin [4,5], chemically [6] or electrochem-
E-mail addresses: jkulys@bchi.lt, juozas.kulys@fm.vtu.lt (J. Kulys). ically [3], whereas the main source of DPPH• is a chemical

0039-9140/$ – see front matter © 2006 Published by Elsevier B.V.


doi:10.1016/j.talanta.2006.11.011

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used without further purification. Recombinant peroxidase


from Coprinus cinereus (rCiP) was received from Novozymes
A/S (Copenhagen, Denmark). The concentration of laccases
and peroxidase was determined spectrophotometrically. The
extinction coefficient for rPpL, was used 7.8 × 104 M−1 cm−1
at 280 nm [13]. The extinction coefficient of rMtL was
1.34 × 105 M−1 cm−1 at 276 nm [14]. The extinction coeffi-
cient for rCiP, was used 1.08 × 105 M−1 cm−1 at 405 nm [15].
The catalase Aspergillus niger was a product of Novozymes
A/S, and the concentration of the enzyme was determined spec-
trophotometrically using absorbance at 280 nm. The extinction
coefficient was assumed 150 mM−1 cm−1 [16]. The solutions
Fig. 1. Structure of Trolox and high reactive laccase substrates. of H2 O2 were prepared from perhydrol (30%) and con-
centration was calculated using an extinction coefficient of
39.4 M−1 cm−1 at 240 nm. Diammonium salt of 2,2 -azinobis(3-
synthesis [7]. When a sample containing an antioxidant (AH) ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtained
is added into the solution of the ABTS•+ or the DPPH• the from Boehringer Mannheim GmbH (Germany), 6-hydroxy-
amount of the radical scavenged is measured by the decrease of 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was
an absorbance. This decrease of absorbance is compared with purchased from Aldrich, 2-phenoxazin-10-yl-ethanol (PET), 3-
the decrease of absorbance produced by the addition of a known phenoxazin-10-yl-propane-1-sulfonic acid sodium salt (PPSA),
amount of Trolox. TE decolourisation assay is widely used for 3-phenoxazin-10-yl-propionic acid (PPA) were synthesized as
the evaluation of the antioxidant activity of pure compounds, described in [17]. Sodium acetate, acetic and hydrochloric
mixtures of antioxidants, and complex samples such as medic- acid were “chemically pure” and were received from Reachim
inal plants, blood plasma and organic tissue, food or beverages (Moscow, Russia). Methanol (purity 99.9%) was received from
[8,9]. Fluka, the highest purified argon was from Aga (Latvia).
The merit of antioxidant capacity of peroxidase (myoglobin)
based method was simple lag-time assay formed during the 2.2. Apparatus and methods
ABTS•+ formation in presence of an oxidant [4]. The appli-
cation of peroxidase or myoglobin, however, requires hydrogen The spectral measurements were performed by using a
peroxide that itself can react with AH. computer-controlled “Nicolet evolution 300” spectrophotome-
Laccases are classified as polyphenol oxidases, and perform ter (Thermo electron corporation, USA) in 1 cm thermostated
the reduction of oxygen to water while oxidizing the substrate quartz cuvette at (25.0 ± 0.1) ◦ C.
[10]. Laccases show broad substrate specificity and catalyse the The kinetic measurements were performed in 20 mM acetate
oxidation phenol derivatives, inorganic and organic metal com- buffer solution pH 5.5. The formation of ABTS radical cation
plexes and other substrates [10]. It was shown that the ABTS was monitored at 414 nm using extinction coefficient 3.5 ×
and other heterocyclic aromatic compounds are oxidized with 104 M−1 cm−1 [18]. The formation of PET and PPA radical
the rate approaching a diffusion of substrates [11]. For this cation was monitored at 530 nm using extinction coefficient
reason laccases are promising for radical cations production. 1.6 × 104 M−1 cm−1 [11]. The concentration of PPSA radi-
Moreover, making the radical cations does not require any addi- cal cation was measured at 530 nm. The extinction coefficient
tional oxidizers with exception of dissolved oxygen. Laccases determined with peroxidase (1.0 × 10−9 M) and hydrogen
to the best of our knowledge had not been used for antioxidants peroxide (1.0 × 10−4 M), and it was 9.5 × 103 M−1 cm−1 .
determination. During antioxidants determination the reaction mixture
The task of this investigation was to develop a method of contained 1.1 × 10−5 M of ABTS, 1.3 × 10−5 M of PET
antioxidants determination with laccases. As laccase substrates (1.3–2.5) × 10−5 M of PPSA, 1.5 × 10−5 M of PPA (0.6–7.6) ×
ABTS and other high reactive phenoxazine derivatives: 2- 10−6 M of Trolox and 1.0 × 10−9 M of rPpL or 1.0 × 10−8 M
phenoxazin-10-yl-ethanol (PET), 3-phenoxazin-10-yl-propane- of rMtL. The mixture contained 5% (v/v) of methanol. The
1-sulfonic acid (PPSA) and 3-phenoxazin-10-yl-propionic acid concentration of laccase and substrate was chosen to perform
(PPA) were used (Fig. 1). measurements during 3–10 min and to get 0.1–0.2 absorbance
change at desired wavelength in 1 cm optical path length cell.
2. Experimental The reaction started with addition of the enzyme solution. The
concentration of the antioxidants in the real samples was deter-
2.1. Materials mined using instead of Trolox solution the diluted extract of the
samples.
Recombinant forms of laccases Polyporus pinsitus (rPpL) For radical cations titration with Trolox the radical cations
and Myceliophthora thermophila (rMtL) expressed in an were synthesized using peroxidase. The solution of ABTS
Aspergillus oryzae and purified as described in [12–14] were (1.3 × 10−4 M), PET (6 × 10−4 M), PPSA (9 × 10−4 M)
received from Novozymes A/S (Copenhagen, Denmark) and and PPA (9 × 10−4 M) was incubated with 1.0 × 10−4 M of

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hydrogen peroxide and 2.0 × 10−8 M of rCiP during 5 min. The


reaction was stopped by adding 2.0 × 10−8 M of catalase. The
solution of radicals was diluted with buffer solution and fixed
amount of Trolox solution was added. The amount of consumed
radical was calculated after 1 min.

2.3. Preparation of solutions

The solutions of PET and PPA were prepared in methanol


with the subsequent dissolution with deionised water. The con-
centration of methanol was 5% (v/v). The solutions of other
reagents were prepared in deionised water and were diluted to
the desired concentration immediately before the use. For the
removal of oxygen the solutions were bubbled with argon during
5 min.
The extracts of real samples, i.e. date-palm, black raisin,
golden raisin, skin of red grape, dice of red grape, were pre-
pared by incubating 0.6–1.5 g of a sample with 1.2–3.0 ml Fig. 2. Absorbance of radical cation of ABTS (1), PET (2), PPA (3) and PPSA
of methanol during 3 days at anaerobic conditions. The mix- (4) at pH 5.5. Concentrations of ABTS•+ 7.3 × 10−6 M, PET•+ 1.3 × 10−5 M,
ture was permanently mixed with magnetic stirrer at room PPA•+ 1.2 × 10−5 M and PPSA•+ 1.2 × 10−5 M. Inset show the titration graph
of PET with Trolox. Each step corresponds the addition of 6 × 10−7 , 1.2 × 10−6 ,
temperature. The extract was diluted 800–102,400 times with
1.8 × 10−6 , 2.3 × 10−6 and 2.9 × 10−6 M of Trolox.
methanol. For the measurements 100 ␮l of the diluted sample
was added into 1.85 ml of thermostated buffer solution contain-
ing ABTS. The reaction started by addition 50 ␮l of laccase
solution.

2.4. Calculations and modelling

To process the data the programs MathCAD 2001 Profes-


sional and GraFit 3.01 were used. A complex kinetic scheme of
simultaneous substrate and antioxidants oxidation was modelled
by Euler’s method [19]. A standard deviation of the parameters
found, i.e. the concentration of an antioxidant and the kinetic
constants varied between 4 and 11%.

3. Results and discussion

3.1. Reaction of antioxidants with radical cations

The radical cations of ABTS, PET, PPA and PPSA produced Fig. 3. The titration of radical cation of ABTS (), PET (), PPA () and
in presence of peroxidase showed strong absorbance in visible PPSA () with Trolox at pH 5.5.
area of spectrum (Fig. 2).
The radical cations of phenoxazine derivatives (PET, PPA
also may origin this difference. This inconsistency was simply
and PPSA) demonstrated remarkable stability during weeks in
eliminated by calibration with Trolox during TE determination
water solution. The addition of Trolox solution to the solu-
of real samples.
tion of the radical cation followed immediate decrease of
The generation of the radical cation of ABTS in pres-
absorbance in visible area of spectrum. The amount of consumed
ence of 1 nM of rPpL is shown in Fig. 4. The addition
radical cation was linearly proportional to the added Trolox
of Trolox stimulated the appearance of lag-period of the
(Fig. 3).
The calculated consumption of the radical cation of ABTS
Table 1
corresponded almost to 2 mol per 1 mol of Trolox since the slope The parameters of radical cations titration with Trolox at pH 5.5
of linear dependence was 1.91 ± 0.07 (Table 1). The stoichiom-
Radical cation Slope Standard error Correlation coefficient
etry of reaction for radical cations of PET, PPA and PPSA was
more than two (Table 1). The reason of this apparent stoichiom- ABTS•+ 1.91 0.07 0.9981
etry increase is unknown. This can be explained by difference of PET•+ 2.58 0.03 0.9996
PPA•+ 2.32 0.07 0.9982
correct and used for the calculations extinction coefficients. A
PPSA•+ 2.69 0.04 0.9996
more complex reaction scheme of the radical cations with Trolox

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of simultaneous antioxidants and substrates oxidation was built


and the antioxidant concentration was determined by data fitting
of kinetic curves.

3.2. Kinetic scheme of simultaneous antioxidants and


substrates oxidation

ABTS and phenoxazine derivatives used in this work are


oxidized with high rate in presence of laccases [11]. Trolox
and other antioxidants as phenol derivatives were also oxidized
with laccase [20]. Therefore the kinetic scheme of substrates
and Trolox oxidation should include simultaneous oxidation
of both types of substrates. In addition, the reaction of the
radical cation with antioxidant (Eq. (4)) should be consid-
ered. A formal redox potential of Trolox is 0.39 V [21] while
Fig. 4. The kinetics of laccase-catalysed radical cation of ABTS production of ABTS it is 0.66 V [18], and of phenoxazine derivatives
in presence of Trolox. Curves correspond to data fitting following a scheme it is 0.63 V versus NHE [17]. Therefore the reaction of the
of laccase action (Eq. (1)–(4)). About 20 mM acetate buffer solution, pH 5.5, radical cation with antioxidant (Eq. (4)) is exothermic and
1.1 × 10−5 M of ABTS, 1.1 × 10−9 M of rPpL, 25 ◦ C; concentration of Trolox
fast.
0 M (), 1.0 × 10−6 M (), 1.7 × 10−6 M (), and 3.5 × 10−6 M ().
The simples kinetic scheme of laccase-catalysed simultane-
ous substrates oxidation can be written:
kinetics curve (Fig. 4). The period was larger at bigger Trolox
concentration. Laccase(red) + O2 + 4H+ → Laccase(ox) + 2H2 O, k1
The reactivity of other substrates was similar to ABTS. How- (1)
ever, a similar rate of radical cations production was indicated
at 10 times larger thermostable laccase (rMtL) concentration Laccase(ox) + S → Laccase(red) + S•+ , k2 (2)
(Fig. 5). In presence of rMtL Trolox also induced lag-period
that was proportional to the antioxidant concentration. Laccase(ox) + Trolox → Laccase(red) + P, k3 (3)
The appearance of lag-period and a sharp absorbance change S•+ + Trolox → S + P, k4 (4)
is associated with a fast radical cations reaction. In principal
the lag-period can be used for antioxidants concentration deter- where Laccase(red) and Laccase(ox) – reduced and oxidized
mination [4], however, for laccase-catalysed process this is not forms of laccase, S or S•+ – ABTS, PET, PPA and PPSA
correct since: (i) production of radical cation is not linear in or respective radical cation, k1 , k2 , k3 , and k4 – the rate
absence of an antioxidant; (ii) antioxidant reacts with laccase as constants.
well as high reactive laccase substrate. For this reason a scheme The fitting of experimental data in absence of antioxidant
(Trolox) produced k1 and k2 values. Due to high concentra-
tion of oxygen (2.5 × 10−4 M) and large k1 value (106 M−1 s−1
[22]) reaction (Eq. (1)) is fast and the fitting permits to calcu-
late k2 . In presence of an antioxidant three parameters (k3 , k4
and antioxidant concentration) are unknown. The constant k4
corresponding to chemical reaction (Eq. (4)) depends on free
energy of reaction following Marcus theory, and for exother-
mic reactions can be as large as 108 M−1 s−1 [23]. The fitting
of experimental data showed that k4 did not influence sig-
nificantly k3 and calculated antioxidant concentration at k4
>106 M−1 s−1 . Therefore this reaction does not limit a process
and k3 and the antioxidant concentration can be found by data
fitting.
At Trolox concentration (0.9–7.6) × 10−6 the following
parameter were found from the calculations (Table 2). The
results demonstrate that for rPpL the substrate showing the
largest oxidation rate is PET, whereas ABTS and PPA are the
Fig. 5. The kinetics of laccase-catalysed radical cation production of PET in best substrates for thermostable laccase rMtL. Unexpected was
presence of Trolox. Curves correspond to data fitting following a scheme of low reactivity of PPSA with rMtL indicating some specificity
laccase action (Eq. (1)–(4)). About 20 mM acetate buffer solution, pH 5.5,
1.3 × 10−5 M PET, 1.0 × 10−8 M of rMtL, 25 ◦ C; concentration of Trolox 0 M
of laccase. A comparison of k2 and k3 values shows that Trolox
(), 7 × 10−7 M (), 1.4 × 10−6 M (), 2.8 × 10−6 M (), and 6.9 × 10−6 M reactivity is similar or just 2–5 times less in comparison to high
(♦). active substrates.

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Table 2
Kinetic characteristics of laccase-catalysed oxidation of high reactive substrates and Trolox in 20 mM acetate buffer solution pH 5.5 at 25 ◦ C
Substrate csubstrate (M) Laccase claccase (M) k2 (M−1 s−1 ) k3 (M−1 s−1 )

ABTS 1.1 × 10−5 rPpL 1.1 × 10−9 1.4 × 106 6.4 × 105
PET 1.3 × 10−5 rPpL 1.0 × 10−9 3.6 × 106 6.4 × 105
PPA 1.5 × 10−5 rPpL 1.0 × 10−9 2.5 × 106 6.4 × 105
PPSA 1.3 × 10−5 rPpL 1.0 × 10−9 2.0 × 106 6.4 × 105
ABTS 1.4 × 10−5 rMtL 1.0 × 10−8 1.2 × 106 2.2 × 105
PET 1.3 × 10−5 rMtL 1.0 × 10−8 4.3 × 105 2.2 × 105
PPA 1.5 × 10−5 rMtL 1.0 × 10−8 9.5 × 105 2.2 × 105
PPSA 2.3 × 10−5 rMtL 1.0 × 10−8 1.5 × 105 2.2 × 105

Table 3
Trolox determination using laccase-catalysed oxidation of ABTS, PET, PPA and PPSA in 20 mM acetate buffer solution, pH 5.5, 25 ◦ C
Substrate Laccase Trolox added (M) Trolox found (M) Recovery (mean ± S.D.) (%)

ABTS rPpL (0.95–7.6) × 10−6 (1.0–6.1) × 10−6 91 ± 9


ABTS rMtL (1.8–7.3) × 10−6 (1.9–6.3) × 10−6 99 ± 8
PET rPpL (0.91–7.3) × 10−6 (1.0–8.3) × 10−6 114 ± 1
PET rMtL (0.91–7.3) × 10−6 (0.7–6.9) × 10−6 82 ± 7
PPA rPpL (0.91–7.3) × 10−6 (1.2–8.3) × 10−6 124 ± 6
PPA rMtL (0.91–7.3) × 10−6 (0.8–6.4) × 10−6 87 ± 1
PPSA rPpL (0.91–3.6) × 10−6 (0.6–2.6) × 10−6 69 ± 3
PPSA rMtL (0.91–7.3) × 10−6 (0.7–6.7) × 10−6 78 ± 7

Trolox recovery determination was performed by fitting


kinetic curves in absence and in presence of different concentra-
tions of the antioxidants. The results demonstrate that Trolox can
be determined with recovery of 100% at less than 10−6 M con-
centration (Table 3). If ABTS was used the recovery of Trolox
was about 100% for both laccases used.
Trolox recovery varied between 82 and 124% if phenoxazine
derivatives were used.

3.3. Antioxidants determination in real samples

The antioxidant activity (TE) in real samples was determined


by samples treatment with methanol and dilution of the extract
with the same solvent. Three dilutions were used, and standard
deviation of TE was calculated from the measurements of these
samples. A typical kinetic curves of radical cation of ABTS
quenching in presence of the date-palm extract is depicted in
Fig. 6. Fig. 6. The kinetics of laccase-catalysed radical cation of ABTS production in
presence of the date-palm extract. Curves correspond to data fitting following
The largest antioxidant activity was found in raisins and a a scheme of laccase action (Eq. (1)–(4)). About 20 mM acetate buffer solution,
dice of red grape (Table 4). The determined values fitted TE pH 5.5, 1.1 × 10−5 M of ABTS, 1.0 × 10−9 M of rPpL, 25 ◦ C; TE 0 M (),
described in the literature [24]. 3 × 10−7 M (), 6 × 10−7 M (), 1.1 × 10−6 M ().

Table 4 4. Conclusions
Antioxidant activity of real samples
Sample TE per 100 g of sample The experiments performed show that laccases can be used
Date-palm (1.9 ± 0.1) × 102
for antioxidant activity determination with spectrophotometer
Black raisin (3.6 ± 0.3) × 102 in presence of high active chromophoric substrates. The use of
Golden raisin (1.0 ± 0.1) × 103 laccases expands the enzymatic method of antioxidants deter-
Skin of red grape (2.4 ± 0.1) × 103 mination, and can be used if the application of peroxidases
Dice of red grape (5.0 ± 0.5) × 103 is unfavourable. As laccase substrate commercially available
Trolox (3.6 ± 0.4) × 105
ABTS was used. New N-substituted phenoxazine derivatives
TE (mean ± S.D.) was determined using 1.1 × 10−5 M of ABTS and demonstrate versatility of the suggested method. The deter-
1.0 × 10−9 M of rPpL. mined Trolox equivalent of real samples fitted described in

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The optimisation of the method may significantly increase the 333.
[12] P. Schneider, M.B. Caspersen, K. Mondorf, T. Halkier, L.K. Skov, P.R.
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